Mitochondria are vital for aerobic respiration, the regulation of Ca2+ homeostasis, apoptosis, aging, and cancer. The intracellular distribution of mitochondria is adaptable to physiological stresses and changes in cellular activity. This plastic control is believed to be especially important in neurons where mitochondria are enriched at regions of intense energy consumption like synapses. Despite the significance of mitochondria for synaptic function, we still do not understand the molecular mechanisms controlling their delivery and targeting to synapses. A comprehensive understanding is urgently needed since abnormal mitochondrial transport, like abnormal mitochondrial function, is associated with various forms of muscular dystrophy, cardiomyopathy, neuropathy, paraplegia, and neurodegeneration. Our previous work suggests that the evolutionary conserved mitochondrial Rho-like GTPase Miro may act as a mitochondrial sensor that integrates intracellular signals to control long-distance transport of mitochondria. Specifically, loss of Drosophila Miro (dMiro) function prevents mitochondrial transport into axons and dendrites while gain of dMiro function leads to an abnormal accumulation of mitochondria at motor nerve terminals. Together, these results suggest dMiro may control anterograde axonal transport and the distribution of mitochondria to synaptic sites. To further test this hypothesis, we will take advantage of the model system Drosophila and genetically manipulate dMiro and other proteins of the mitochondrial transport machinery. Mutant effects on mitochondrial transport will be primarily examined in larval motor neurons, their axons and axon terminals by live imaging of GFP-tagged mitochondria to resolve the following key issues: Aim 1 will resolve whether dMiro promotes net-anterograde axonal transport by increasing the efficiency of microtubules (MT) plus end- or decreasing minus end-directed transport. Aim 2 will determine the role of dMiro's EF-hand Ca2+ binding domains for mitochondrial transport and/or the intracellular distribution of mitochondria. Aim 3 will test the molecular mechanisms by which dMiro may control mitochondrial transport. The proposed project is expected to reveal important molecular signaling mechanisms that regulate the long- distance transport of mitochondria and their use-dependent distribution into synaptic terminals. Uncovering these signaling pathways will significantly expand our understanding of basic mechanisms and accelerate the development of new concepts for detecting, treating, and/or preventing disorders that are caused by defective mitochondrial transport pathways and/or impaired mitochondrial function.